1. Field of the Invention
This invention relates to a method for quantitative analysis of an analyte in a liquid sample using an analytical element. More particularly, this invention relates to an improvement in a method for quantitatively analyzing an analyte in a liquid sample by applying the liquid sample on one of a set of analytical elements and measuring a color developed in the analytical element through reflection photometory in which said set of analytical elements are the same elements as a standard element except for deviation of sensitivity to the analyte, but said deviation of sensitivity is essentially equivalent to each other among the set of analytical elements, the calibration curve of the standard element with respect to said analyte being predetermined.
2. Description of prior arts
As a method for quantitative analysis of an analyte (i.e., a substance to be analyzed) in a liquid sample, there has been generally employed a reaction-in-solution system involving a homogeneous reaction in a dilute solution and a procedure of measuring a light transmitted through the solution. This system gives analytical data with high accuracy, so long as the system is conducted by a skilled operator using a long period of time.
In the field of clinical tests, however, an analytical system which can be operated more simply and more rapidly is strongly requested by people concerned, such as medical doctors.
In answer to the above-mentioned request, a dry analytical system using an analytical element in the form of a sheet containing a dry reagent responsive to the analyte has been proposed to replace the above analytical system involving the reaction in a solution. In particular, a multilayer analytical element comprising a number of laminated layers in the element has been paid attention.
In the dry analytical system, the quantitative determination of an analyte in a liquid sample is performed by applying the liquid sample of the analytical element and measuring a color developed in the element through reflection photometry. It is known that the relationship between a content of an analyte in an applied liquid sample and the optical reflection density value observed on the color development due to the application of the liquid sample does not follow Beer's law, while the relationship between a content of an analyte in a liquid sample and the transmittance value observed on the color development in a solution due to the introduction of the liquid sample does follow such law. Accordingly, in the case that the reflection photometry is employed in the analysis, a calibration curve which shows the relationship between a content of an analyte in an applied liquid sample and the optical reflection density to be observed on the color development due to the application of the liquid sample is ought to be predetermined for each kind of analytical element.
Generally, the same calibration curve is applicable to all analytical systems involving the same analyte and analytical elements belonging to the same nature, for example, those having the same composition. However, in practice the compositions of the analytical elements slightly vary depending upon the conditions of their preparations. Otherwise, even if analytical elements having the almost the same compositions are prepared, the analytical elements are apt to be denatured slighty in the course of their storage under influence of light, heat, humidity and so on. Accordingly, if a precise quantitative determination is intended, it is preferred to prepare a calibration curve on each set of the analytical elements, in which the analytical elements in one set are considered to have essentially the same composition and the same sensitivity to the analyte because these elements were prepared under the same conditions and have been stored under the same conditions. The procedure of the preparation of a calibration curve for each set of analytical elements is time-consuming and accordingly is not advantageous in the practical operation.
As is described in the above, the quantitative analysis based on the reaction-in-solution, namely wet system, follows the Beer's law. Accordingly, it is known that a calibration curve determined on a standard analytical element is well applicable with simple adjustment to an analytical element which is deviated with respect to the composition and sensitivity in the course of its preparation and/or its storage. For instance, Japanese Patent Provisional Publication No. 58(1983)-109837 teaches a method for adjusting a predetermined calibration curve which comprises a step of measuring an absorbance for at least one sample having a known concentration and a step of adjusting the calibration curve based on the measured absorbance and the absorbance corresponding to the sample having the same concentration, the latter absorbance being obtained using the predetermined calibration curve. This adjusting method is very simple but is only applicable to a measuring system based on measurement of transmitted light.
In more detail, since the measuring system utilizing transmission of light follows the Beer's law, the relationship between the concentration or content of an analyte in a sample and the optical transmission density can be expressed by a linear equation. This means that such relationship can be expressed by a straight line in a graph. Accordingly, the deviation of the sensitivity of the transmittance-measuring system can be easily adjusted by such simple method.
In contrast, such simple adjusting method is not applicable to the measuring system based on reflection photometry, because the relationship between the concentration or content of an analyte in a sample and the optical reflection density is expressed not by a linear equation but by a very complicated equation. This means that such relationship can be expressed not by a straight line but by a curve in a graph. An example of such curve is illustrated in the attached. FIG. 1. In FIG. 1, a calibration curve for a standard analytical element for glucose analysis in the form of an integral multilayer element which indicates a relationship between a glucose content in serum and an optical reflection density value is illustrated by the solid curve. The dotted curve in FIG. 1 shows a calibration curve for an analytical element which is deviated in the sensitivity to glucose from the standard element. As is understood from the curves, it is very difficult to determine the relationship between the calibration curve for the standard analytical element and the calibration curve for the deviated analytical element. Accordingly, the deviation of the sensitivity of the measuring system based on reflection photometry cannot be adjusted by the above-mentioned simple method disclosed in the prior art.
As to the relationship between the transmittance and reflectance in photographic color prints, F. C. Williams and F. R. Clapper have proposed in Journal of the Optical Society of America, Vol. 43, No. 7, 595-599(July, 1953) a conversion equation, but the proposed conversion equation is extremely complicated using a number of parameters. Accordingly, it is not easy to utilize the proposed conversion equation in analytical systems to be performed in practice.